01_110_04_Luciana%20Rego-Oliveira%20e%20Ricardo%20de%20Faria_In%20vitro%20propagation

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Acta Scientiarum. Agronomy Maringá, v. 27, no. 1, p. 1-5, Jan./March, 2005

In vitro propagation of Brazilian orchids using traditional culture

media and commercial fertilizers formulations

Luciana do Valle Rego-Oliveira e Ricardo Tadeu de Faria*

1Departamento de Agronomia, Universidade Estadual de Londrina, C. P. 6001, 86051-990, Londrina, Paraná, Brasil. *Author for

correspondence. e-mail: [email protected]

ABSTRACT. Some species of Brazilian orchids are practically extinct in their natural

habitats. Large scale propagation of in vitro native orchids is a way of preserving them.

Specific culture media for some species have already been described, but there are no

studies on Catasetum fimbriatum and Cyrtopodium paranaensis. The aim of the present

study was to establish in vitro propagation protocol of C. fimbriatum and C. paranaensisto reintroduce these species in natural habitat. The traditional culture media were: MS; MS

modified with ½ macronutrients; MS modified with ¼ macronutrients, Vacin & Went and

Knudson C. The commercial fertilizers formulations were: N.P.K (10-5-5) 2 mL L-1 and

formulation N.P.K (10-30-20) 3 g L-1. The data, assessed six months after the beginning of

the experiment, consisted of: number of roots, main root length, pseudobulb diameter,

plant dry weight, root dry weight and plant height. The best results of C. fimbriatum plant

development were obtained using MS medium complete and MS medium modified with

half of macronutrients for canopy length. For the number of roots, the best results were

obtained using MS modified with half of macronutrients. The best results for C.

paranaensis plant development were obtained using MS modified with half of the

macronutrients for canopy length and MS modified with 1/4 of macronutrients for number

of roots.

Key words: tissue culture, culture media, comercial fertilizers, Catasetum fimbriatum, Cyrtopodium

paranaensis, orchids propagation.

RESUMO. Propagação in vitro de orquideas brasileiras utilizando meios de cultura

tradicionais e formulações com fertilizantes comerciais. Algumas espécies de orquídeas

brasileiras encontram-se praticamente extintas no seu habitat natural. Para que isto não

ocorra, a propagação in vitro em larga escala é uma maneira de preservá-las. O

estabelecimento de meios de cultura específicos para algumas espécies já foram descritos,

porém não existem estudos para Catasetum fimbriatum e Cyrtopodium paranaensis. O

objetivo deste estudo foi o estabelecimento de um protocolo de propagação in vitro para C.

fimbriatum e C. paranaensis para a re-introdução das mesmas em seus habitats naturais. Os

meios de cultura tradicionais utilizados foram: MS; MS modificado com ½ dos

macronutrientes; MS modificado com ¼ dos macronutrientes, Vacin & Went e Knudson C.

Os adubos comerciais utilizados foram: formulação N.P.K (10-5-5) 2 mL L-1 e formulação

N.P.K (10-30-20) 3 g L-1. Os dados avaliados após seis meses do início do experimento

foram: número de raízes, comprimento da raiz principal, largura de bulbo, peso seco da parte

aérea , peso seco das raízes e comprimento da parte aérea. Os melhores resultados para o

desenvolvimento vegetativo e enraizamento de C. fimbriatum foram os meios MS com total e

½ dos macronutrientes para desenvolvimento da parte aérea, já para número de raízes o

melhor meio de cultura foi o MS modificado com ½ dos macronutrientes. Para C.

paranaensis o melhor resultado para o desenvolvimento vegetativo foi o apresentado no meio

de cultura MS modificado com ½ dos macronutrientes e para número de raízes foi o MS

modificado com ¼ dos macronutrientes.

Palavras-chave: cultura de tecidos, meio de cultura, fertilizantes comerciais, Catasetum fimbriatum,

Cyrtopodium paranaensis, propagação de orquídeas.

Introduction

The Orchidaceae family is one of the largest of

the Angiosperm families, consisting of about 700

genera and 35,000 species distributed throughout the

world. Orchids are found in all the states of Brazil,

and in greater quantity in the Mata Atlantica (Atlantic



2 Rego-Oliveira e Faria


Forest), which goes from Pernambuco state to Rio

Grande do Sul . These plants are also found in

abundance in riverside forest throughout the country,

in the Savannah of Central Brazil, in the Amazon

forest and even in the Northeastern tropical thorn bush forest (caatinga). There are more than 3,500

different Brazilian species, and the second major

number of species is found in Colombia (Miller and

Warren, 1996).

There are more than 50 species of Catasetum in

the world and also a great variety of hybrids, where

the flower can vary greatly in size and color. It is a

tropical plant and generally easy to grow (Hersh,

1996). The Cyrtopodium paranaensis orchid is a

tropical climate species typical of Brazilian

hillsides. The Cyrtopodium genus is found in the

Americas from Florida (USA) to Argentina

(Meneses, 1995a, b).

There are few studies on the most suitable culture

media for sowing native Brazilian orchids, and there

is no information on the best maturity point of seeds

and their sensitivity to sterilization and

acclimatization processes (Stancato and Faria, 1996).

Interest in Brazilian orchid species has increased

sharply both in the domestic and external markets but

the propagation of these plants has been

unsuccessful, because there is little data available on

their cultivation (Kerbauy, 1995).

Orchids have an immense biochemical,

physiological and genetic diversity and there is arange of culture media designated for the

multiplication of each species. The Knudson C

(1946) is the most used medium for in vitro sowing

and the formulas described by Vacin and Went

(1949) and Murashige and Skoog (1962) are the most

used for clonal propagation (Arditti e Ernest, 1990).

An ideal nutritive medium supplies the essential

substances for tissue growth and controls the standard

of in vitro development to a large extent. Because of

this, several organic compounds are added to the

culture medium to complement the substances

biosynthesized by the cells and to supply the

metabolic, energetic and structural requirements of the cell (Caldas et al ., 1998).

Orchids can be rapidly multiplied by in vitro

culture from sprout tips or meristems from

Cymbidium orchids (Morel, 1960). This technique

was used for several genera and found almost

immediate practical application at the beginning of

the 1970s. Currently, many commercial laboratories

in Europe, North America and Southeast Asia

annually produce millions of low cost of orchid

plantlets using this methodology (Bornman, 1993).

In the last years, a large number of research about

orchids culture media, specially with Catasetum

fimbriatum were published (Peres et al., 1999; Peres

e Kerbauy, 1999; Majerowicz et al., 2000).

The aim of the present study was to establish in

vitro propagation protocol of C. fimbriatum and C.

paranaensis to reintroduce these species in natural

habitat.

Material and methods

The two Brazilian orchid species used in this

study were Catasetum fimbriatum (Morren) Lindl.

and Cyrtopodium paranaensis Schlt. The capsules of

orchids with seeds inside were disinfected with 1.5%

active sodium hypochlorite for 15 minutes. After

disinfection, the capsules were washed in autoclaved

water and opened with a scalpel. The seeds inside the

capsule were collected and inoculated in the Knudson

C germination medium within a blade flow chamber.

After seed germination, during the protocormformation phase, 15 protocorms were inoculated per

flask with the following treatments: T1) MS (1962);

T2) MS modified with ½ macronutrients; T3) MS

modified with ¼ macronutrients; T4) Vacin and

Went (1949); T5) Knudson C (1946); T6)

Commercial formulation N.P.K (10-5-5) 2 mL L-1;

T7) Commercial formulation N.P.K (10-30-20) 3 g

L-1

. In all treatments 30 g L-1

of sucrose and 6 g L-1

of agar were added. In treatments T6 and T7, they

were prepared with commercial formulations easily

found in the market. These traditional culture media

could be easily used in Bio-facture, as they are not

difficult to be found, they do not cost a lot and because of the facility of its preparation when

compared with the traditional ones.

Flasks of 250 mL capacity were used, with 50 mL

of autoclaved culture medium each. The pH of each

culture media was adjusted to 6.0 before putting in

autoclave. These flasks were kept in a controlled

environment at 25-27ºC and 16 hours light from a

fluorescent lamp. The following characteristics were

analyzed: root number, length of largest root, pseudo

bulb diameter, canopy dry weight, root dry weight

and canopy length. The assessments were made six

months after the beginning of the experiment.

A complete randomized block design was used

and there were five replications containing 15

protocorms per replication. The data obtained were

submitted to analysis of variance and of the Tukey

test.

Results and discussion

There were differences among the several culture

media in vitro cultivation of C. fimbriatum for root

number, length of largest root (cm), pseudo bulb

diameter (cm), plant dry weight (mg), root dry weight

(mg) and plant height (cm). The plants with the

greatest growth of C. fimbriatum were observed in



In vitro propagation of Brazilian orchids 3


treatments T1 (MS) and T2 (MS modified with ½ the

macronutrients), both with 6.00 cm. The plants with

lowest growth were observed in treatment T6

(N.P.K.- 10-5-5), with 1.80 cm (Table 1).

Table 1. Mean values for plant height, number of roots, largest root

length, peudobulb diameter, plant and root dry weight of

Catasetum fimbriatum six months after the beginning of the

experiment. Universidade Estadual de Londrina, State of Paraná,

2003.

Treatments Plant

height

(cm)

Noof

roots

Main root

length

(cm)

Psuedobulb

diameter

(cm)

Plant dry

weight

(mg)

Root dry

weight

(mg)

T1 MS 6.00 a1

2.24 b 5.00 c 0.25 c 26.20 bc 25.00 c

T2 MS with ½

macronutrients

6.00 a 3.50 a 7.00 b 0.61 a 61.00 a 54.20 a

T3 MS with ¼

macronutrients

3.30 c 2.12 bc 3.30 d 0.50 b 24.80 c 13.00 d

T4 Vacin and Went 3.74 bc 1.60 c 1.52 e 0.20 c 7.60 dc 3.00 e

T5 Knudson C 4.00 b 2.20 b 2.54 d 0.53 ab 46.20 ab 13.00 d

T6 N.P.K (10-5-5) 1.80 d 1.60 c 8.04 a 0.25 c 1.40 d 34.00 b

T7 N.P.K (10-30-20) 3.40 c 3.24 a 3.00 d 0.50 b 23.00 c 24.20 cC.V % 7.14 12.80 11.70 10.90 28.53 18.50

DMS 0.60 0.60 1.00 0.10 21.00 9.001 means followed by the same letter on the vertical do not differ by the Tukey test at 5%

probability.

The plants with the greatest root number in C.

fimbriatum were found in treatments T2 (MS

modified with ½ the macronutrients) and T7 (N.P.K.-

10-30-20) with 3.50 and 3.24, respectively. The

lowest root number were found in treatments T4

(Vacin and Went) and T6 (N.P.K.- 10-5-5), both with

1.60 (Table 1).

Peres et al. (1999) proposed that an endogenous

balance from auxin to cytokinins is important toshoot formation in C . fimbriatum.

The plants with most satisfying root length were

observed in T6 (N.P.K- 10-5-5) with 8.04 cm and the

least in T4 (Vacin and Went) with 1.52 cm. The

plants that show the greatest pseudo bulb diameter

were those kept in treatment T2 (MS with ½

macronutrients) with 0.61 cm. Plants that show the

least pseudo bulb diameter were those kept in

treatments T1 (MS), T4 (Vacin and Went) and T6

(N.P.K.- 10-5-5) with 0.25, 0.20 and 0.25 cm,

respectively (Table 1).

Culture media differed in the in vitro cultivation

of C. paranaensis for number of roots, length of thelargest root (cm), plant dry weight (mg), root dry

weight (mg) and plant height (cm) (Table 2).

Characteristic pseudo bulb diameter was not analyzed

for C. paranaensis because they do not differ

significantly.

Table 2. Mean values for plant height, number of roots, largest root

length, plant and root dry weight, for Cyrtopodium paranaensis, in

the different culture media 6 months after the beginning of the

experiment. Universidade Estadual de Londrina, State of Paraná,

2003.

Treatments Plant height

(cm)

Noof roots Main root

length (cm)

Plant dry

weight (mg)

Root dry

weight (mg)

T1 MS 6.64 b1 2.80 d 1.50 d 30.40 a 20.40 bc

T2 MS with ½

macronutrients

8.70 a 2.76 d 1.80 cd 28.20 a 16.40 c

T3 MS with ¼

macronutrients

5.10 c 4.76 a 3.60 b 17.0 b 41.40 ab

T4 Vacin and Went 3.75 d 1.92 e 1.00 e 10.80 c 25.20 bc

T5 Knudson 3.19 d 2.60 d 2.02 c 14.00 bc 15.20 c

T6 N.P.K. (10-5-5) 1.91 e 4.00 b 4.30 a 16.80 b 53.60 aT7 N.P.K. (10-30-20) 3.70 d 3.40 c 3.80 b 19.00 b 50.00 a

C.V. % 11.73 8.31 6.71 13.28 28.26

DMS 1.11 0.52 0.34 5.20 4.401 means followed by the same letter on the vertical do not differ by the Tukey test at 5%

probability.

The plants having the greatest growth of C.

paranaensis were observed in treatment T2 (MS

modified with ½ macronutrients), with 8.70 cm. The

plant with the lowest growth was observed in

treatment T6, NPK (10-5-5), with 1.91 cm. The

plants with the greatest root number in C.

paranaensis were found in treatment T3 (MS

modified with ¼ the macronutrients) with 4.76, while

the lowest root number were found in treatment T4(Vacin and Went) with 1.92 (Table 2). The plants

with the most satisfying root length (cm) were

observed in treatment T6 (NPK 10-5-5) with 4.30 cm,

while plants that show the least were found in

treatment T4 (Vacin and Went) with 1.00 cm.

Collins and Dixon (1992) cultivated the Diuris

longifoli orchid in vitro in Vacin and Went culture

(1949) and observed that the plantlets had excellent

growth. However, in this study the orchids grew

less in the same culture medium mentioned above.

The most adequate means presented for plant

dry weight for C. paranaensis were for treatments

T1 (MS), T2 (MS modified with ½ macronutrients)

with 30.4 and 28.2 mg, respectively, and the least

mean was for treatment T4 (Vacin and Went) with

10.8 mg. The greatest means for root dry weight

were found in treatments T6 (N.P.K.- 10-5-5) and

T7 (N.P.K.- 10-30-20) with 53.6 and 50.0 mg,

respectively, and the least means were for

treatments T2 (MS modified with ½ macronutrients)

and T5 (Knudson C) with 16.4 and 15.2 mg,

respectively.

The best growth culture media for Laelia

cinnabarina Batem. orchid was MS medium

modified with ½ macronutrients and completemedium Hoagland and Arnon (1950). However, more

vigorous plantlets were obtained in the MS medium

modified with ½ the macronutrients (Stancato and

Faria, 1996).

When the two orchids C. fimbriatum and C.

paranaensis were compared to the number of roots,

there were differences among the culture media. The

treatment with greatest root formation induction for

C. fimbriatum was T2 (MS modified with ½

macronutrients) with 3.50 whereas for C.

paranaensis the T3 (MS modified with ¼

macronutrients) was the best with 4.76.

The two orchids, C. fimbriaum and C.



4 Rego-Oliveira e Faria


paraneensis were different in plant height. The best

treatments for C. fimbriatum were T1 (complete MS)

and T2 (MS modified with ½ macronutrients), both

with 6.00 cm. However for Cyrtopodium paranaensis

the best treatment was T2 (MS modified with ½ themacronutrients) with 8.7 cm.

Kraus and Kerbauy (1992) compared different

culture media for protocorm formation by roots

explants of Catasetum pileatum orchid roots. The

Murashige and Skoog (1962), Vacin and Went

(1949) and Knudson C (1946) culture media were

assessed and the best culture medium for protocorm

regeneration was Murashige and Skoog. Majerowicz

et al (2000) suggest that organic nitrogen and NH4+

are probably the most important nitrogen sources to

C. fimbriatum plants.

The traditional MS media culture was more

appropriate for the propagation in vitro of the two

studied orchids when compared to the formulations

containing the commercial fertilizers. This happened

because the MS culture media is rich in macro and

micronutrients while the commercial fertilizers have

only N-P-K.

These results confirm that suitable culture media

should be determined for each species and using

more appropriate nutritive formulas can optimize the

plant quality produced in vitro.

Acknowledgement

We are grateful to Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq)

for financial support.

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Received on April 29, 2004.

Accepted on December 08, 2004.

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